Process for preparing a pharmaceutical formulation of contrast agents

ABSTRACT

The invention relates to a process for preparing a liquid pharmaceutical formulation containing a complex of macrocyclic chelate with a lanthanide and a mol/mol amount of free macrocyclic chelate of between 0.002% and 0.4%, advantageously between 0.02% and 0.3% and very advantageously between 0.025% and 0.25%, the macrocyclic chelate advantageously being chosen from DOTA, NOTA, DOTAGA, DO3A, BT-DO3A, HP-DO3A and PCTA, and is preferably DOTA, the said process comprising the following successive steps:
         a) determination of a theoretical target concentration of free macrocyclic chelate C tcl  in the final liquid pharmaceutical formulation;   b) preparation of a liquid pharmaceutical composition containing, firstly, the complex of macrocyclic chelate with a lanthanide, and, secondly, free macrocyclic chelate and/or free lanthanide;   c) measurement in the pharmaceutical formulation obtained in step b) of the concentration of free macrocyclic chelate C cl  and/or of free lanthanide C ll ;   d) adjustment of C cl  and/or of C ll  so as to obtain C cl =C tcl  and C ll =0.

The invention relates to pharmaceutical formulations of contrast agents,in particular of complexes of chelates with paramagnetic metal ions,especially for magnetic resonance imaging, and to industrially efficientprocesses for obtaining these formulations.

Many contrast agents based on complexes of chelates with lanthanides(paramagnetic metal), in particular with gadolinium, are known, and aredescribed, for example, in document U.S. Pat. No. 4,647,447. Severalproducts are marketed, especially based on macrocyclic chelates such asDOTA gadoterate (1,4,7,10-tetraazacyclo-dodecane-N,N′,N″,N′″-tetraaceticacid) and gadoteridol HPDO3A, and linear chelates such as DTPA(diethylenetriaminepentaacetic acid) and DTPA-BMA (gadodiamide).

In the body, the complexes of chelates with lanthanide are in asituation of chemical equilibrium, which may lead to a risk of undesiredrelease of the lanthanide, and more especially of gadolinium. A personskilled in the art is thus led to seek technical solutions that limitthis risk in order to solve the complex problem of tolerance in thepatient as safely as possible. This problem is all the more difficultsince the administration of contrast agents is often repeated duringdiagnostic examinations and/or for the guiding and monitoring of theefficacy of a therapeutic treatment.

Several approaches for improving the tolerance of complexes of chelateswith gadolinium are described in the prior art. More than twenty yearsago (U.S. Pat. No. 5,876,695), those skilled in the art were working onformulations consisting of the addition to a lanthanide-complexingchelate of an amount of free chelate, i.e. non-complexed chelate. Thisexcess chelate is intended to compensate for an undesired release oflanthanide, the excess chelate then complexing with the releasedlanthanide (Gd³⁺ metal ion). U.S. Pat. No. 5,876,695 in particulardescribes an excess of free linear chelate, in particular of DTPA.Documents EP 450 078, U.S. Pat. No. 5,876,695 and US 2004/0 170 566especially describe the use of salts of these chelates (especially ofcalcium, sodium, zinc or magnesium).

According to another very different approach, which is recalled indocument WO 2007/106 544, a person skilled in the art grafted onto thechelates chemical groups intended to increase the affinity of thechelate for the metal.

Despite all these prior-art studies, the complex problem of tolerancestill exists, especially in situations at risk of more pronouncedtolerance for the administration of MRI contrast products.

A new problem has moreover recently appeared in the matter of tolerance,namely a pathology known as NSF (nephrologic systemic fibrosis, orfibrogenic dermopathy), which may be at least partly correlated to theexistence of free gadolinium, i.e. non-complexed gadolinium, in thebody. This disease has led to health authorities being alerted asregards certain categories of patients with respect to marketedgadolinium-based contrast agents.

In summary, this problem of tolerance of complexes of chelates withlanthanides remains complex and important.

The Applicant has worked on the specific case of macrocyclic chelates,and has demonstrated, for these chelates, the advantage of using anamount of free chelate, which is particularly advantageous in terms oftolerance.

The Applicant has shown that with macrocyclic chelates, and inparticular DOTA, results in terms of tolerance are very advantageous,using a very low excess of free chelate, more specifically between 0.02%and 0.4% and in particular between 0.025% and 0.25%.

Consequently, in view of this low amount, a new problem arises, which isunknown in the prior art, namely the need for extremely precise anddelicate industrial-scale control of the concentrations of free chelateand thus of the manufacture of the product to arrive at this range oftarget values of amount of free chelate, these values needing to bestable, including after storage for several months or years.

Specifically, taking into account the production volumes, which are ofthe order of several tens of tons of active principle, the Applicant hadto develop a preparation process that makes it possible to ensure thereliability and reproducibility of the composition of commercializedbatches. In particular, the Applicant found that the mixing ofstoichiometric amounts on the basis of the theoretical calculation doesnot sufficiently satisfactorily give at the industrial scale therespective amounts of complex of chelate with the lanthanide and of freechelate in low concentration in the pharmaceutical formulation. Thereason for this is that it is then necessary to perform several analysissteps, which takes several hours, and significantly increases theindustrial cost price of the product. In contrast, the Applicant'sprocess makes it possible especially to prepare beforehand and tooptimize the analytical device, which is important as regards its impacton the quality of the final product.

More specifically, by respecting the stoichiometric proportions and byadding an excess of DOTA intended not to be complexed with thelanthanide, it is not possible at the industrial scale to achievesufficiently reproducibly in the final pharmaceutical solution an excessof free DOTA in the target range, especially given:

-   -   1) the uncertainty of weighing at the industrial scale, which        does not make it possible to correctly ensure the ratio (of the        order of 1000) between the chelate and the excess chelate, given        the small amount of excess chelate;    -   2) the variability of the hygroscopic characteristics of the        chelate (associated with its acid functions).

It is pointed out, specifically, that to prepare an industrial amount,typically, for example 200 litres of a 0.5 M solution of gadoliniumchelate (for example DOTA-Gd), the amount of DOTA to be added in excessafter complexation of the DOTA with the lanthanide, to obtain an excessof free DOTA of 0.1 mol/mol %, would be about 40 g of DOTA in 200 litresof the DOTA solution (40 g in addition to the 40 kg of DOTA initiallyplaced in solution), which does not allow sufficiently reliablereproducibility at the industrial level.

This problem has been solved by the Applicant by means of using at leastone step of measuring in the liquid pharmaceutical formulationconcentrations of free macrocyclic chelate (C_(cl)) and/or of freelanthanide (C_(ll)) and at least one step of adjusting the C_(cl) and/orthe C_(ll) so as to obtain the desired concentration of C_(cl) andC_(ll)=0, advantageously by modifying the amounts of macrocyclic chelateor of lanthanide in the pharmaceutical composition.

Throughout the application, the equality C_(ll)=0 is used to define thatC_(ll) in the formulation injected into the patient is zero orsubstantially zero (typically less than 10⁻¹⁰ M and advantageously lessthan 10⁻² M or 10⁻¹⁴ M), the possible presence in solution of anextremely small amount of lanthanide not being able to be totallyexcluded. The reason for this is that concentrations less than 10⁻¹⁰ Mcannot be measured sufficiently reliably by the current analyticalmethods.

It is recalled herein that in document U.S. Pat. No. 5,876,695 for thelinear DTPA chelate, in Example 2, the amounts of excess chelate aredefined from the start on the calculated basis of the stoichiometry, andwithout a step of prior adjustment or measurement of the concentrations.More specifically, in the said example, 0.5 mol of DTPA and 0.25 mol ofgadolinium oxide (Gd₂O₃) are mixed in accordance with the stoichiometricproportions, and a 0.1 mol/mol % (0.5 mmol) excess of ligand is added;this does not ensure the target amount of ligand desired by theApplicant in the case of a large-scale industrial manufacture.

Thus, according to one aspect, the present invention relates to aprocess for preparing a liquid pharmaceutical formulation of complex ofmacrocyclic chelate with lanthanide, the said process comprising atleast one step of measuring in the liquid pharmaceutical formulationconcentrations of free macrocyclic chelate (C_(cl)) and/or of freelanthanide (C_(ll)) and at least one step of adjusting the C_(cl) and/orthe C_(1l), so as to obtain (sufficiently stably in the finalpharmaceutical solution, i.e. the pharmaceutical formulation intended tobe administered to the patient) a mol/mol amount of free macrocyclicchelate of between 0.002% and 0.4%, advantageously between 0.02% and0.3% and very advantageously between 0.025% and 0.25%.

The present invention thus relates to a process for preparing a liquidpharmaceutical formulation containing a complex of macrocyclic chelatewith a lanthanide and a mol/mol amount of free macrocyclic chelate ofbetween 0.002% and 0.4%, advantageously between 0.02% and 0.3% and veryadvantageously between 0.025% and 0.25%, the macrocyclic chelateadvantageously being chosen from DOTA, NOTA, DOTAGA, DO3A, BT-DO3A(gadobutrol), HP-DO3A and PCTA, and is advantageously DOTA, the saidprocess comprising the following successive steps:

-   -   a) determination of a theoretical target concentration of free        macrocyclic chelate C_(tcl) in the final liquid pharmaceutical        formulation;    -   b) preparation of a liquid pharmaceutical composition        containing, firstly, the complex of macrocyclic chelate with a        lanthanide, and, secondly, free macrocyclic chelate and/or free        lanthanide;    -   c) measurement in the pharmaceutical formulation obtained in        step b) of the concentration of free macrocyclic chelate C_(cl)        and/or of free lanthanide C_(ll);    -   d) adjustment of C_(cl) and/or of C_(ll) so as to obtain        C_(cl)=C_(tcl) and C_(ll)=0.

For the purposes of the present invention, the term “amount of freemacrocyclic chelate” means the proportion of free macrocyclic chelaterelative to the amount of complexed macrocyclic chelate (gadoteric acidin the case of DOTA-Gd) present in the formulation in mol/mol. In therest of the description, it will be referred to without preference asthe “amount of free macrocyclic chelate” or the “excess free macrocyclicchelate”.

For the purposes of the present invention, the term “free macrocyclicchelate” means any macrocyclic chelate not complexed with lanthanide.

For the purposes of the present invention, the term “free lanthanide”means any lanthanide not complexed with a macrocyclic chelate.

As device for analysis/assay of the free macrocyclic chelate, anysuitable equipment is used. Advantageously, a potentiometer or capillaryelectrophoresis is used for the macrocyclic chelate. More specifically,in the presence of copper sulfate, the free DOTA contained in thesolution obtained from the complexation step (in bulk) complexes thecopper. The excess copper sulfate is assayed in pH 5 buffered medium, bypotentiometry, with a solution of EDTA in the presence of acopper-indicating electrode and a reference electrode.

The analysis/assay of the free lanthanide is performed by using, forexample, a solution of EDTA in the presence of xylenol orange Arsenezoas turning-point indicator.

In order to perform step d), several solutions are possible as afunction of the C_(ll) and C_(cl) measured in step c).

In particular, the following solutions are concerned:

-   -   if C_(ll)>0 and/or C_(cl)<C_(tcl), the adjustment may        advantageously be performed by adding free macrocyclic chelate        and/or by eliminating free lanthanide and/or by modifying the pH        as described hereinbelow;    -   if C_(ll)=0 and C_(cl)>C_(tcl), the adjustment may        advantageously be performed by eliminating free macrocyclic        chelate and/or by adding free lanthanide and/or by modifying the        pH as described hereinbelow.

Advantageously, the elimination of free lanthanide is performed bypassing through an ion-exchange resin. It is thus possible, for example,to use a resin of styrene/divinylbenzene copolymers which containsiminodiacetate ions acting as chelating group for the binding with themetal ions (Gd³⁺ in particular).

Advantageously, the elimination of free macrocyclic chelate isperformed, for example, by filtration, advantageously using resins (forexample anionic resins).

In one particular embodiment, step b) consists in mixing a solution offree macrocyclic chelate (initial) and of free lanthanide (initial) soas to obtain complexation of the lanthanide by the macrocyclic chelate.

The lanthanide is advantageously added in the form of oxide (gadoliniumoxide in particular), but the invention also covers other possible formsof lanthanide, especially the lanthanide salts known to those skilled inthe art.

The precise experimental conditions of step b) are detailed in theexamples. Advantageously, the temperature for step b) is between 60 and100° C., and is advantageously about 80° C. Advantageously, thepharmaceutical formulation is then cooled before the adjustment step d).The duration of step b) is, for example, from 1 hour to 3 hours.

Moreover, throughout the description hereinabove and hereinbelow of theadjustment variants of step d), it is understood that the complexationstep b) may be performed in several sub-steps which would be equivalentto an overall complexation step. The complexation may be performed, forexample, by preparing about half the final volume of the tank, and thenadding gadolinium oxide at acidic pH.

It is possible to use a preparation process such that:

-   -   in step b), the amounts of free macrocyclic chelate (initial)        and of free lanthanide (initial) added are equal to the        stoichiometric proportions;    -   step c) consists in measuring C_(cl) and/or C_(ll);    -   step d) consists in adding to the formulation obtained in        step b) the amount of free macrocyclic chelate necessary,        firstly, to complete, if necessary the complexation of the free        lanthanide and to obtain C_(ll)=0 (or substantially equal to 0),        and, secondly, to obtain C_(cl)=C_(tcl).

For the purposes of the present invention, the expression “the amountsof free macrocyclic chelate and of free lanthanide added are equal tothe stoichiometric proportions” means that the amounts added are suchthat, in the light of the stoichiometry of the complexation reaction,all the lanthanide and all the chelate should be in complex form andthere should be no free macrocyclic chelate.

However, advantageously, it is preferred, in the process according tothe invention, for step c) of measurement of the concentrations to beperformed in a medium in which the complexation reaction of step b) isperformed:

-   -   by using a difference between the stoichiometric proportions and        the amounts of free lanthanide and of free macrocyclic chelate        added in step b),    -   or by modifying the pH to shift the chemical equilibria in        favour or in disfavour of complexation.

For the purposes of the present invention, the expression “differencebetween the stoichiometric proportions and the amounts of freelanthanide and of free macrocyclic chelate added in step b)” means thatthe amounts of free lanthanide and of free chelate added in step b) aresuch that, in the light of the stoichiometry of the complexationreaction, not all the lanthanide is complexed by the chelate (excesslanthanide and/or deficit of chelate relative to the stoichiometry) ornot all the chelate is complexed with the lanthanide (excess chelateand/or deficit of lanthanide).

Advantageously, this difference is such that the macrocyclicchelate/lanthanide or lanthanide/macrocyclic chelate mol/mol ratio isless than or equal to 1.4, advantageously between 1.001 and 1.3,particularly advantageously between 1.005 and 1.2, and in particularbetween 1.005 and 1.02. It is also pointed out that this ratio may beadapted depending on whether an excess of chelate or an excess oflanthanide is used. When an excess of lanthanide is used for thecomplexation, advantageously the lanthanide/macrocyclic chelate mol/molratio is typically less than or equal to 1.2. When an excess of chelateis used for the complexation, the macrocyclic chelate/lanthanide mol/molratio is advantageously less than or equal to 1.4.

Thus, in one advantageous embodiment, the amounts of free macrocyclicchelate and of free lanthanide added are such that not all themacrocyclic chelate is complexed with the lanthanide or such that notall the lanthanide is complexed with the macrocyclic chelate.Consequently, after this step b), the pharmaceutical formulation willtypically comprise macrocyclic chelate-lanthanide complex and:

-   -   either free macrocyclic chelate,    -   or free lanthanide.

In this case, the preparation process according to the present inventionis characterized in that, in step b), there is a difference between theamounts of free macrocyclic chelate and of free lanthanide added and thestoichiometric proportions, this difference advantageously being suchthat the macrocyclic chelate/lanthanide or lanthanide/macrocyclicchelate mol/mol ratio is less than or equal to 1.4, advantageouslybetween 1.001 and 1.3, particularly advantageously between 1.005 and 1.2and in particular between 1.005 and 1.02.

According to particular embodiments, the ratio will be, for example,1.01, 1.02, 1.03 or 1.04. This gives, for example, the concentrationspresented in Table 1 below, which shows the case of an excess of initialfree lanthanide, given that an excess of initial chelate may also beused.

Concentration of free Concentration of free macrocyclic chelatelanthanide (initial) (1) (initial) added in step b) added in step b)Lanthanide/ (M) (M) chelate ratio 0.480 0.520 1.083 0.487 0.513 1.0530.492 0.508 1.032 0.497 0.504 1.014 (1) this is the amount of Gd³⁺, andnot of Gd₂O₃

For example, in the case of the chelate DOTA, an amount of free chelatecorresponding to a concentration of 0.497 M of chelate and an amount offree lanthanide corresponding to a concentration of 0.504 M oflanthanide, which corresponds to a lanthanide/DOTA mol/mol ratio ofx=1.014 (with x=0.504/0.497), will be added in step b).

Another way of expressing this difference relative to the stoichiometricproportions is to define it relative to the lanthanide concentration inthe final solution.

In the case of this example which illustrates an excess of lanthanide,the difference is 0.6%=100*[(0.5-0.497)/0.5)], for a formulation at 0.5M of gadolinium at stoichiometry. The difference is thus, for example,advantageously between 0.1 mol % and 2 mol % of the concentration atstoichiometry of the pharmaceutical formulation.

In one embodiment that is also advantageous (preferred mode) theadjustment step d) is performed without touching the total amount oflanthanide present in the formulation, i.e. without adding or removingany lanthanide. In this case, only the total amount of macrocyclicchelate and/or the pH is modified.

For the purposes of the present invention, the term “total amount oflanthanide” means all the lanthanide present in free form and incomplexed form.

For the purposes of the present invention, the term “total amount ofmacrocyclic chelate” means all the macrocyclic chelate present in freeform and in complexed form.

Thus, in a first case (case A of the preferred mode), an excess oflanthanide relative to the macrocyclic chelate is added in step b) andstep d) consists in adding free macrocyclic chelate.

In this case, the preparation process according to the present inventionis characterized in that:

-   -   in step b), the amounts of free macrocyclic chelate and of free        lanthanide added are such that not all the lanthanide is        complexed, the lanthanide/macrocyclic chelate ratio (mol/mol)        advantageously being less than 1.2;    -   step c) consists in solely measuring of C_(ll), C_(cl) typically        being equal to 0 (or substantially equal to 0);    -   step d) consists in adding to the formulation obtained in        step b) the amount of free macrocyclic chelate necessary,        firstly, to complete the complexation of the free lanthanide so        as to obtain C_(ll)=0 and, secondly, to obtain C_(cl)=C_(tcl).

In a second case (case B of the preferred mode), an excess ofmacrocyclic chelate relative to the lanthanide is added in step b). Inthis case, depending on the excess of chelate, step d) consists inadding or removing macrocyclic chelate.

Specifically, when the excess chelate added in step b) makes it possibleto obtain C_(cl)<C_(tcl), it is then appropriate in step d) to addfurther free macrocyclic chelate in order to obtain C_(cl)=C_(tcl).

On the other hand, if the excess chelate added in step b) makes itpossible to obtain C_(cl)>C_(tcl), it is then appropriate in step d) toremove free macrocyclic chelate (where appropriate, to add free Gd) inorder to obtain C_(cl)=C_(tcl).

In this latter case, the preparation process according to the presentinvention is characterized in that:

-   -   in step b), the amounts of free macrocyclic chelate and free        lanthanide added are such that all the lanthanide is complexed        and that C_(cl)>C_(tcl), the macrocyclic chelate/lanthanide        ratio (mol/mol) advantageously being less than 1,2;    -   step c) consists in solely measuring C_(cl), C_(ll) being equal        to 0;    -   step d) consists in removing the appropriate amount of free        macrocyclic chelate so as to obtain C_(cl)=C_(tcl).

It is pointed out that, in cases A) and B), as illustrated in thedetailed Example 2, the adjustment step d) comprises at the end a stepof adjustment of the pH and of the volume, advantageously with megluminefor DOTA.

In a third case (case C) the pH of the formulation (and optionally otherfunctionally equivalent chemical parameters) is controlled so as toshift the reaction equilibrium in order to obtain at the end the targetpharmaceutical solutions (excess amount of target ligand).

According to one embodiment, the preparation process is such that:

-   -   in step b), the amounts of free macrocyclic chelate and of free        lanthanide added are equal to the stoichiometric proportions;    -   it comprises between steps b) and c) an intermediate step b1) of        modifying the pH of the pharmaceutical formulation obtained in        step b) so as to shift the chemical equilibria in favour or in        disfavour of complexation;    -   step c) is performed on the formulation obtained in step b1);    -   step d) consists in adjusting C_(cl) and/or C_(ll) so as to        obtain C_(cl)=C_(tcl) and C_(ll)=0 (or substantially 0) by        modifying the pH so as to shift the equilibrium in the direction        opposite to that of step (b1) and optionally by adding or        removing free macrocyclic chelate.

For example, the process according to the invention is characterized inthat:

-   -   in step b), the mixing is performed at a pH of between 4 and 7,        advantageously between 5 and 6.5,    -   step b1) consists in increasing the pH using a base up to a        value of between 10 and 13 and advantageously between 11 and 12,    -   step d) consists in lowering the pH down to a value of between        6.5 and 7.5 and advantageously between 6.8 and 7.4, and        optionally adding or removing free macrocyclic chelate.

For example, the complexation is performed at a pH below 6 (for examplebetween 3 and 6 and advantageously between 5 and 6) and the pH is thenraised, for example, to about 12 (for example with NaOH), and the pH isthen adjusted to about 7.

In variants of the process without pH adjustment, as described in thedetailed Example 2 of the present patent application, in step 2, thecomplexation is typically performed at a pH below 6 (for example between3 and 6), the pH being brought directly to about 7. Without going intothe complex mechanisms, it is indicated that, given the thermodynamicconstants varying according to the pH, step b1) of the process with pHadjustment is such that it makes it possible to achieve the target rangeof excess free chelate in the pharmaceutical solution at least up to theexpiry of the shelf life of the pharmaceutical solution. Increasing thepH makes it possible to shift the equilibrium in the direction from anexcess of macrocyclic chelate to a level substantially equal to thetarget excess amount. Next, by reducing the pH, a reduction at a verylow rate in the amount of free macrocyclic chelate is obtained suchthat, over the shelf life of the product, the amount of freelanthanide/macrocyclic chelate does not change unfavourably. This wouldresult from the implied thermodynamic constants associated with the pHmodifications.

It may also be pointed out that when the free lanthanide measurementwill be performed (at pH 7), the concentration will be lower than if thepH change had not been made, and the adjustment is then made with thecorrect amount of chelate.

Furthermore, without going into the detail of the complexationmechanisms that take place at the molecular level in several phases(described especially in Chem. Eur. J., 2004, 10, 5218-5232), theApplicant points out that it was not at all obvious that the processwith adjustment would make it possible to obtain this result.

In another advantageous embodiment, step b) consists in preparing asolid complex [chelate-lanthanide] and in dissolving it (in water).

In this case, step d) is performed on a liquid formulation obtained bydissolving a solid [chelate-lanthanide] complex.

According to this embodiment, step b) of the process according to theinvention comprises two sub-steps:

-   -   i) the preparation of a solid complex [chelate-lanthanide] and    -   ii) the dissolution of the complex obtained in step i).

The adjustment in step d) may be performed as described previously indetail (addition of chelate or of lanthanide, removal of chelate or oflanthanide, adjustment by pH modification).

The preparation of the solid complex, which is advantageouslycrystalline [chelate-lanthanide], involves, where appropriate, at leastone treatment step (filtration, concentration, crystallization, drying,spraying, etc.) for obtaining the appropriate physicochemicalcharacteristics, especially in terms of solubility and purity.

In addition, in another particularly advantageous embodiment, a furthertechnical problem was solved by the Applicant for the industrialmanufacture of a pharmaceutical formulation of contrast agent based on amacrocyclic chelate-lanthanide complex, while at the same time making itpossible to maximize the tolerance profile of the contrast product. Morespecifically, contrary to the prior-art teaching, for example U.S. Pat.No. 5,876,695, which suggests the presence of large quantities ofcalcium (introduced in the form of calcium chelate) in thepharmaceutical formulation, the Applicant has demonstrated that, in thecase of the process according to the present invention, a very lowamount of calcium would make it possible to ensure the industrialcontrol of this process and to obtain a very well-tolerated product.

More specifically, the reliability of step c) of measuring the amountsof chelate and/or of lanthanide with common industrial analytical toolsis markedly improved when the amount of calcium in the components used(in particular in the macrocyclic chelate, the lanthanide and the waterused in step b)) is less than a very low target value of around 15 to200 ppm. The amount of calcium (quantity of calcium) in the macrocyclicchelate used in step b) is advantageously less than 200 ppm andadvantageously in the region of or less than 15 ppm. For example, if theamount of calcium in the DOTA (active principle in the form of powdersupplemented with water in step b)—see the detailed Example2—dissolution step 1) is too high (and especially greater than 200 ppm),calcium may complex the chelate and the adjustment of the amount of freechelate will not be performed sufficiently satisfactorily.

The low amount of calcium in the pharmaceutical solution makes itpossible to avoid possible disadvantageous interferences regarding theassays of free macrocyclic chelate (for example by complexing thecalcium with the chelate) and thus to obtain an assay of the freechelate and its adjustment in a manner that is particularly effectivefor manufacture at the industrial scale at the required high level ofquality. Furthermore, a very low calcium concentration controlled in thefinal product administered to the patient (especially the meglumine saltof gadolinium DOTA) is advantageous as regards the calcaemia of thepatients in so far as it makes it possible to avoid any homeostasisimbalance: the impact of the injected product (typically at a dose ofless than 20 ml) on the calcaemia is at most in the region of 0.5%. Theamount of calcium in the administered contrast product is advantageouslyless than 50 ppm and especially less than 20 ppm, for example between 1and 5 ppm. For example, for the meglumine salt of gadolinium DOTA, alimit of 15 μg of Ca/g of DOTA powder (15 ppm) used in step b)corresponds to 3 μg Ca/ml of liquid contrast product administered to thepatient (there is about 0.202 g of DOTA per ml of administered liquidcontrast product), i.e. 3 ppm in this contrast product.

The different variants of the process according to the invention asdescribed previously thus advantageously comprise, before the measuringand adjustment steps c) and d), an intermediate step b2) of controllingthe amount of calcium in the formulations obtained in step b).

Where appropriate, in particular if the amount of calcium in the finalsolution is greater than 15 ppm or advantageously greater than 10 ppm,this intermediate step comprises, following this control, the removal ofthe excess calcium.

Thus, according to one aspect, the process according to the presentinvention is characterized in that the amount of calcium in the liquidpharmaceutical formulation administered to the patient is less than 50ppm, especially less than 20 ppm, and preferably less than 5 ppm, theprocess advantageously comprising, before step c), an intermediate stepb2) of measuring the amount of calcium and, where appropriate, ofremoving the excess calcium.

Furthermore, the different variants of the process according to theinvention as described previously advantageously comprise, before stepb), control of the amount of calcium in the components used in step b),and especially in the macrocyclic chelate intended to be dissolved, inthe lanthanide (typically used in oxide form), and in the water.Advantageously, the amount of calcium in these components is less than150 ppm and preferably less than 15 ppm.

Very advantageously, the Applicant has succeeded in removing the excesscalcium in the chelate (powder) used in step b), by means, in particularfor DOTA, of a purification by crystallization using a water-ethanolmixture, which makes it possible to obtain an amount of calciumadvantageously less than 50 ppm. The water used for step b) is alsoadvantageously purified, where appropriate by means of a suitabletreatment, for example descaling with acids to prevent any undesiredamount of calcium.

A gadolinium oxide with a purity very close to 100%, substantially of99.999%, will preferably be used in particular.

Furthermore, it will be preferred to check that the meglumine used atthe end of the adjustment step d) also comprises a small amount ofcalcium.

The process is also advantageously such that it uses components thathave extremely low amounts of metals (for example nickel and aluminium)liable to interact with the chelate, disrupting the assays. Thus, theprocess advantageously includes a step of checking the amount of thesemetals before the measuring and adjustment steps b) and/or c) and/or d).

Finally, the process according to the present invention alsoadvantageously comprises an additional step e) of checking C_(cl) andC_(ll), irrespective of the variant described above.

The process according to the present invention is, according to onepreferred embodiment, characterized in that the pharmaceuticalformulation is a pharmaceutical formulation of meglumine salt of theDOTA-gadolinium complex.

The Applicant's process makes it possible to obtain the targetformulations safely. This process makes it possible to solve the problemrepresented by the in situ complexation, in a pharmaceuticalmanufacturing reactor (into which is added the pharmaceuticalformulation agent). Specifically, when the lanthanide is Gd³⁺, megluminewill be used as formulation agent. However, given the physicochemicalcharacteristics of gadoteric acid, the mixing of the three components(powder of non-complexed chelate, lanthanide powder and megluminepowder) in the same reactor would not be sufficiently satisfactory.Thus, the process according to the invention that allows this problem tobe solved consists in engaging the complexation, measuring thedifference relative to the target, and adjusting.

Overall, the Applicant's process thus makes it possible to incorporatethe chelation process into the pharmaceutical production, with anadvantage especially in terms of cost price and quality.

In one advantageous embodiment, an agent for blocking the freelanthanide, other than the free macrocyclic chelate, is added in stepb). Advantageously, this blocking agent is a polycarboxylic acid,especially a dicarboxylic, tricarboxylic or tetracarboxylic acid, inparticular a citrate or a derivative thereof.

As regards the general inventive concept of the target range of amountof free macrocyclic chelate (0.002% to 0.4%, advantageously 0.02% to0.3% and in particular 0.025% to 0.25%), the Applicant points out thatthis range differs from the teaching of patent U.S. Pat. No. 5,876,695illustrated in particular by its examples, at least for the followingreasons.

The Applicant's target range is very narrow, and corresponds to aselection within the very broad range presented in the said document.

The formulations described in U.S. Pat. No. 5,876,695, which concernmacrocyclic chelates (especially Examples 3 and 4), are formulationswith salts of chelate (calcium disodium, zinc disodium DOTA) and notwith free chelate. The amounts of excess salts therein are moreover veryhigh, at least 10%. However, in the present patent application, only thefree chelates are used, and not in the form of salts.

The formulations presented in U.S. Pat. No. 5,876,695, which have anamount of free chelate of about 0.1%, concern only linear chelates(DTPA), and the DTPA formulation at 0.08% described is clearly indicatedas a control solution, the said document suggesting, on the contrary,the use of a much higher amount, 2% or quite probably more.

Specifically, the only test presented as regards tolerance on the use ofchelates, in Table 2 and in column 6 (lines 62-67) of the said document,shows that the reduction in toxicity is markedly less favourable for anamount of free chelate of 0.08 mol/mol % (Formulation A for which theamount is established on the basis of the ratio between 0.5 mmol Gd DTPAand 0.0004 mmol DTPA/kg), in comparison with the 2% amount correspondingto the advantageous formulation (Formulation B for which the amountestablished on the basis of the ratio between 0.5 mmol Gd DTPA and 0.01mmol DTPA/kg) and which is described as a low value (column 6, line 61).

The Applicant thus worked on formulations with an amount of freemacrocyclic chelate about 5 to 100 times lower than that explicitlyrecommended by document U.S. Pat. No. 5,876,695. It was thusdemonstrated by the Applicant, surprisingly, that macrocyclic chelates,and more especially DOTA, behave differently from linear chelates suchas DTPA as regards tolerance, resulting from the presence of an excessof free chelate.

More specifically, whereas the tolerance appears to be improved withDTPA by increasing the excess free chelate from 0.08% to 2%, thetolerance degrades, in contrast, for DOTA by increasing the excess freechelate, passing from very low values (0.025% to 0.25%) to a value of2%. Consequently, the transposition of values to reduce the risk oftoxicity, between a linear chelate (in particular DTPA), and macrocyclicchelates (in particular DOTA), is not at all evident. This is moreoverwhat is illustrated by the current complex discussions in the scientificcommunity in the context of NSFs with regard to the tens of millions ofdoses of contrast agents already injected in man, discussions on thesubject of the complexation kinetics and/or on comparisons of structuresbetween chelates.

To this end, according to another aspect, the invention relates to apharmaceutical formulation that may be obtained via the processaccording to the present invention, characterized in that it containsbetween 0.002 and 0.4 mol/mol %, more especially between 0.02 and 0.3mol/mol % and very advantageously between 0.025 and 0.25 mol/mol %, offree macrocyclic chelate, advantageously of free DOTA.

By virtue of the adopted selection of the range of excess freemacrocyclic chelate, in particular of free DOTA, a value of freelanthanide in solution, and in particular of gadolinium, of about from10⁻¹⁰ M to 10⁻¹⁴ M at physiological pH, is obtained.

The concentration of complexed chelate in the formulation is typicallybetween 1 μM and 1 M, with an administered dose of about from 0.01 to 5mmol/kg. The concentration of the injected formulation is typicallyabout 0.5 M.

The process particularly advantageously relates to the preparation ofthe pharmaceutical formulation of the meglumine salt of theDOTA-gadolinium complex: the macrocyclic chelate and the freemacrocyclic chelate are DOTA, the lanthanide is gadolinium, and theprepared salt is the meglumine salt.

Advantageously, the pharmaceutical formulation according to the presentinvention is characterized in that the macrocyclic chelate is DOTA andin that the formulation contains between 0.02 and 0.08 mol/mol % of freeDOTA.

This lower range is liable to have several physiological advantages:

-   -   limiting a risk of chelation for certain diseases of endogenous        cations (for example zinc or copper) by the presence of an        overly large excess of macrocyclic chelate,    -   limiting the inhibition of metalloenzymes, especially ACE, with        an impact on the regulation of arterial hypertension, for        example,    -   avoiding unfavourable medicinal interactions with metallic        active principles:    -   lithium, bismuth, platinum, etc.,    -   avoiding disrupting the seric dosages of endogenous metals,    -   avoiding medicinal interactions with active principles that are        complexing, for example detoxifying (deferroxamine, cyclam,        etc.).

In another advantageous embodiment, the pharmaceutical formulationaccording to the present invention is characterized in that themacrocyclic chelate is DOTA and in that the formulation contains between0.15 and 0.25 mol/mol % of an excess amount of free DOTA.

This higher range is liable to have several physiological advantages:

-   -   optimally limiting the amount of free gadolinium injected, the        free gadolinium being a toxicity risk and possibly being        involved in phagocytosis mechanisms associated with certain        diseases,    -   minimizing the in vivo transmetallation in pathological        situations, especially transmetallation by iron (increase in        seric iron).

This higher range is also an advantage for further improving thestability of the formulation to be injected over time (dechelation underunsuitable storage conditions: heat, depressurization in aircraft,excessive exposure to light, etc.).

According to one embodiment, the amount of free macrocyclic chelate isbetween 0.09% and 0.15%. This median range is liable to combineadvantages of the lower and higher ranges.

The choice of the amount of free macrocyclic chelate may be optimized inparticular as a function of the risk of the patients for variouspathologies or pathological risks associated with the mechanismspresented hereinabove. For example, in the case of patients presenting arisk of NSF, an excess of macrocyclic chelate in the median or highrange may be preferred, to minimize any release of gadolinium.

Very low values of excess free chelate are, however, also liable to havea beneficial effect in the pathology NSF if it turns out in certaincategories of patients (kidney failure patients in particular) that thispathology is partly associated with a presence of free chelate, whichwould involve in vivo transmetallation or similar phenomena that areunfavourable in terms of tolerance.

According to another aspect, the calcium content of the pharmaceuticalformulation (administered to the patient) according to the invention isless than 50 ppm, advantageously less than 30 ppm and advantageouslyless than 15 ppm.

According to another aspect, the invention relates to use of a contrastproduct formulation, the said formulation comprising a complex ofmacrocyclic chelate with a paramagnetic metal ion and an amount of freemacrocyclic chelate of between 0.025% and 0.25%, advantageously of aformulation according to the present invention, for improvement of thetolerance.

According to another aspect, the invention relates to a method forimproving the in vivo tolerance of an MRI contrast product based onmacrocyclic chelate, and more especially on DOTA, which consists inusing an excess of free chelate in an amount of between 0.025 and 0.25mol/mol %, especially 0.025-0.08%, 0.09-0.15%, 0.16-0.25%.

Advantageously, the concentration of chelate (complexed chelate) in theformulation is between 0.5 and 0.9 M.

The macrocyclic chelate that is useful in the context of the presentinvention is advantageously chosen from the following chelates: DOTA,NOTA, DO3A, BT-DO3A, HPDO3A, PCTA, DOTAGA and derivatives thereof, andis most particularly DOTA. The chemical formulae of these chelates arewidely known to those skilled in the art, and are recalled, for example,in WO 2007/042 504, on pages 20 to 23, and WO 2003/011 115, on pages 8to 11.

The invention also relates to the use of a pharmaceutical formulationaccording to the invention for the preparation of a diagnosticcomposition for medical imaging, or for diagnostic monitoring of theefficacy of a therapeutic treatment, and to a diagnostic methodcomprising the administration of a pharmaceutically acceptable amount ofa formulation according to the invention.

For diagnosis in MRI, the intravenous administration by injectionusually as a saline solution is typically performed at a dose of from 1to 500 μmol Gd/kg. The pharmaceutically acceptable unit doses willdepend on the nature of the chelate, the route of administration, and onthe patient and especially on the nature of the disorder to be studied.For an intravenous injection and observation by magnetic resonance, theconcentration of the solution will typically be between 0.001 and 0.5mol/litre, and from 0.001 to 0.1 millimol/kg will be administered to thepatient, depending on the case. Higher clinical doses may also bepractised, for example a triple dose (0.3 millimol/kg).

Among the advantageous diagnostic indications, mention will be made ofthe indications already used clinically, and the indications for whichthe results are improved by virtue of the formulations according to theinvention. Mention will thus be made of the following indications andimprovements thereof: angiography, cerebral imaging, vascular imaging,imaging of cardiovascular, cancer, neurodegenerative and inflammatorypathologies, any indication with perfusion imaging, any indicationcombining the use of several contrast products, especially MRI, X-rayscanner, SPECT, PET, PET CT, and any indication with successiveadministrations of contrast products or in multimodal imaging.

According to embodiments, these novel formulations may be chosen to beadministered in combination with or in place of prior-art formulationsas a function of the diagnostic profile of the patient, and especiallyof the profile of tolerance of the patient to the contrast products.

An installation comprising a device for evaluating the tolerance of thepatient, and a device for administering the formulation of the contrastproduct as a function of the result given by the evaluation device maythus be used. Several risks may be evaluated, especially the risk of NSF(nephrogenic fibrosis). Where appropriate, the MRI product isco-administered simultaneously with or subsequently to at least oneanti-NSF therapeutic agent (anti-fibrosis agent known therapeutically,especially steroids, anti-inflammatories or vitamins, for example).

Where appropriate, an evaluation of the patient's risk with respect toNSF is performed to optimize the dose of injected contrast product (forexample, the dose may be reduced relative to the common clinical dose,if it makes it possible, while avoiding any risk, to obtain sufficientlysatisfactory information to obtain the signal in imaging). At least oneanti-NSF therapeutic agent, especially an anti-inflammatory, steroid orvitamin, may be co-administered.

To further reduce the risk of toxicity of the lanthanide in the case ofat-risk patients, the Applicant also studied formulations comprising:

-   -   as in the prior art: the chelate of lanthanide (for example        gadoteric acid: DOTA-Gd complex) and the salification agent for        neutralizing the chelate, for example meglumine (organic base),    -   but in addition with at least one biocompatible excess blocking        agent, intended to block any lanthanide (Gd³⁺) that might        otherwise remain free in the formulation.

Among the blocking agents that will especially be used are organicanions such as monocarboxylic or polycarboxylic acids (advantageouslytricarboxylic or tetracarboxylic, such as citrate and derivativesthereof), hydroxy acids, or other agents capable of an advantageouscoordination interaction with the lanthanide.

The blocking agent may thus be introduced into the formulation and/orco-administered to the patient.

DETAILED EXAMPLES 1) Example 1 In Vivo Tolerance

The tolerance results in Table 2 (acute toxicity in mice for adiagnostic solution of DOTA) show that formulations containing from0.025 to 0.25 mol/mol % of free macrocyclic chelate are three times lesstoxic than the formulation close to 2%.

Free DOTA Male LD₅₀ Female LD₅₀ Test mol/mol % Mmol/kg Mmol/kg 1 0.0512.41 13.59 2 0.09 13.06 13.50 3 0.25 12.02 12.07 4 1.98 4.80 4.80

2) Example 2 Process for Preparing Formulations of Lanthanide ChelateMixture of a Solution of Chelate and of a Solution of Lanthanide

The preparation of formulations in which the macrocyclic chelate is DOTAis more specifically described. Table 3 below gives an example of theamounts used for the manufacture of a solution of 100 litres of DOTA(industrial amount).

Component Amount DOTA (1) 20.100 kg (i.e. 0.497 M) Gadolinium oxide(expressed as 9.135 kg (i.e. 0.504 M) anhydrous product) Meglumine(expressed as anhydrous 9.215 kg product) Solution for adjusting DOTA to5% qs 15-35 mg per 100 ml amount of free DOTA 3 N meglumine solution qspH = 6.8-7.4 at 20° C. Injection-grade water qs 100 litres (1)1,4,7,10-Tetraazacyclododecane-N,N′,N″,N′′′-tetraacetic acid

Step 1: Dissolution

40 litres of injection-grade water at 80° C. are placed in a 100-litremanufacturing tank, the injection of nitrogen is started, and the 20.100kg of DOTA and the 9.135 kg of gadolinium oxide are then incorporatedwith stirring. The complexation is performed at a pH below 6, forexample between 3 and 6, for example at pH 4. The gadolinium oxide inthe presence of DOTA forms a water-soluble acid complex.

Step 2: Measurements

After step 1, a sample is taken and the free gadolinium is assayed.

Step 3: Adjustment of the Free Species

The adjustment of the solution is advantageously performed withgadolinium oxide or DOTA.

A DOTA-adjusting solution is thus added qs an amount of 15-35 mg per 100ml.

Step 4: Cooling

The final solution from step 3 is cooled to 30° C., for example bycirculating cold water in the tank jacket.

Step 5: Adjustment of the pH and of the Mass Per Unit Volume

The acid function of the complex formed is salified with meglumine andthe pH at 20° C. is adjusted to 6.8-7.4. The concentration is adjustedby adding injection-grade water.

The following are thus introduced into the manufacturing tank:

-   -   9.125 kg of meglumine    -   and a solution of meglumine pH=6.8-7.4 at 3N    -   injection-grade water, qs.

The final solution is then filtered and then placed in bottles typicallysterilized by autoclaving.

3) Example 3 Process for Preparing Formulations of Lanthanide ChelateDissolution of a Solid Complex [Chelate-Lanthanide]

This example illustrates the manufacture of a small amount of product,the appropriate transposition being performed at the industrial scale.

Int. 1 Int. 2 (Gd₂O₃) Int. 3 M_(w) (g · mol⁻¹) 404.42 362.70 580.63 m(g) 10 4.48 n (mol) 0.025 (1 Eq) 0.0125 (0.5 Eq)

10 g (0.025 mol; 1 eq) of macrocyclic chelate DOTA are dissolved in 200ml of water by heating to 80° C., in a three-necked flask equipped witha condenser, a thermometer and a pH meter. The measured pH is 3.7. It isadjusted to 6 with 2N NaOH solution. 4.48 g (0.0125 mol; 0.5 eq) ofgadolinium oxide are added. The pH is readjusted and kept stable atbetween 6 and 7 by adding 1N HCl. The reaction is left at 80° C. withstirring.

The residual free gadolinium is removed by means of a chelex resinprerinsed with water. To do this, the reaction mixture is brought to pH5 (the resin is more efficient). The whole is left for 2 hours withstirring at room temperature. The pH rises to between 6.5 and 7. Theresin is removed by filtration.

The complex is precipitated in ethanol to remove the salts (5 volumes ofEtOH per 1 volume of water).

An assay of the salts is performed by titration with a 0.05N silvernitrate solution. Quantification of the free gadolinium is alsoperformed by calorimetric assay with Arsenazo (III). 11.5 g of productare obtained (white powder).

Yield=80%; HPLC purity: 98%; LC/MS (ES+mode): z=1 (m/z=559).

The dissolution in water is then performed via suitable methods, forexample using a water at 45° C., with stirring for about 30 minutes, andwith adjustment of the pH.

1. Process for preparing a liquid pharmaceutical formulation containinga complex of macrocyclic chelate with a lanthanide and a mol/mol amountof free macrocyclic chelate of between 0.002% and 0.4%, the said processcomprising the following successive steps: a) determination of atheoretical target concentration of free macrocyclic chelate C_(tcl) inthe final liquid pharmaceutical formulation; b) preparation of a liquidpharmaceutical composition containing, firstly, the complex ofmacrocyclic chelate with a lanthanide, and, secondly, free macrocyclicchelate and/or free lanthanide; c) measurement in the pharmaceuticalformulation obtained in step b) of the concentration of free macrocyclicchelate C_(cl) and/or of free lanthanide C_(ll); d) adjustment of C_(cl)and/or of C_(ll) so as to obtain C_(cl)=C_(tcl) and C_(ll)=0. 2.Preparation process according to claim 1, characterized in that step b)consists in mixing a solution of free macrocyclic chelate and of freelanthanide so as to obtain complexation of the lanthanide by themacrocyclic chelate.
 3. Preparation process according to claim 2,characterized in that, in step b), there is a difference between theamounts of free macrocyclic chelate and of free lanthanide added and thestoichiometric proportions.
 4. Process according to claim 3,characterized in that the difference between the amounts of freemacrocyclic chelate and of free lanthanide added and the stoichiometricproportions is such that the macrocyclic chelate/lanthanide orlanthanide/macrocyclic chelate mol/mol ratio is less than or equal to1.4.
 5. Preparation process according to claim 3, characterized in that:in step b) the amounts of free macrocyclic chelate and of freelanthanide added are such that not all the lanthanide is complexed; stepc) consists in measuring C_(ll), C_(cl) being equal to 0; step d)consists in adding to the formulation obtained in step b) the amount offree macrocyclic chelate necessary, firstly, to complete thecomplexation of the free lanthanide so as to obtain C_(ll)=0, and,secondly, to obtain C_(cl)=C_(tcl).
 6. Process according to claim 5,characterized in that, in step b), the lanthanide/macrocyclic chelateratio (mol/mol) is less than 1.2.
 7. Preparation process according toclaim 3, characterized in that: in step b), the amounts of freemacrocyclic chelate and of free lanthanide added are such that all thelanthanide is complexed and that C_(cl)>C_(tcl); step c) consists inmeasuring C_(cl), C_(ll) being equal to 0; step d) consists in removingthe appropriate amount of free macrocyclic chelate so as to obtainC_(cl)=C_(tcl).
 8. Process according to claim 7, characterized in that,in step b), the macrocyclic chelate/lanthanide ratio (mol/mol) is lessthan 1.2.
 9. Preparation process according to claim 1, characterized inthat: in step b), the amounts of free macrocyclic chelate and of freelanthanide added are equal to the stoichiometric proportions; step c)consists in measuring C_(cl) and/or C_(ll); step d) consists in addingto the formulation obtained in step b) the amount of free macrocyclicchelate necessary, firstly, to complete, if necessary, the complexationof the free lanthanide and to obtain C_(ll)=0, and, secondly, to obtainC_(cl)=C_(tcl).
 10. Preparation process according to claim 1,characterized in that: in step b), the amounts of free macrocyclicchelate and of free lanthanide added are equal to the stoichiometricproportions; it comprises between steps b) and c) an intermediate stepb1) of modifying the pH of the pharmaceutical formulation obtained instep b) so as to shift the chemical equilibria in favour or in disfavourof complexation; step c) is performed on the formulation obtained instep b1); step d) consists in adjusting C_(cl) and/or C_(ll) so as toobtain C_(cl)=C_(tcl) and C_(ll)=0 by modifying the pH so as to shiftthe equilibrium in the direction opposite to that of step (b1) andoptionally by adding or removing free macrocyclic chelate.
 11. Processaccording to claim 10, characterized in that: in step b), the mixing isperformed at a pH of between 4 and 7, step b1) consists in increasingthe pH using a base up to a value of between 10 and 13, step d) consistsin lowering the pH down to a value of between 6.5 and 7.5, andoptionally adding or removing free macrocyclic chelate.
 12. Processaccording to claim 1, characterized in that step b) consists inpreparing a solid complex [chelate-lanthanide] and in dissolving it. 13.Process according to claim 1, characterized in that the amount ofcalcium in the liquid pharmaceutical formulation administered to thepatient is less than 50 ppm.
 14. Process according to claim 13,characterized in that it comprises, before step c), an intermediate stepb2) of measuring the amount of calcium and, where appropriate, ofremoving the excess calcium.
 15. Process according to claim 1,characterized in that it comprises an additional step e) of checkingC_(cl) and C_(ll).
 16. Process according to claim 1, characterized inthat the macrocyclic chelate is advantageously chosen from DOTA, NOTA,DOTAGA, DO3A, BT-DO3A, HP-DO3A and PCTA.
 17. Process according to claim16, characterized in that the macrocyclic chelate is DOTA.
 18. Processaccording to claim 17, characterized in that the pharmaceuticalformulation is a pharmaceutical formulation of meglumine salt of theDOTA-gadolinium complex.
 19. Process according to claim 1, characterizedin that an agent for blocking the free lanthanide is added in step b).20. Process according to claim 19, characterized in that the agent forblocking the free lanthanide is a polycarboxylic acid. 21.Pharmaceutical formulation that may be obtained via a process accordingto claim 1, characterized in that it contains between 0.002 and 0.4mol/mol % of free macrocyclic chelate.
 22. Formulation according toclaim 21, characterized in that the free macrocyclic chelate is freeDOTA.
 23. Formulation according to claim 21, characterized in that itcontains between 0.02 and 0.3 mol/mol % of free macrocyclic chelate. 24.Formulation according to claim 21, characterized in that it containsbetween 0.025 and 0.25 mol/mol % of free macrocyclic chelate. 25.Formulation according to claim 22, characterized in that it containsbetween 0.02 and 0.08 mol/mol % of free DOTA.
 26. Formulation accordingto claim 22, characterized in that it contains between 0.15 and 0.25mol/mol % of free DOTA.
 27. Formulation according to claim 21,characterized in that its calcium content is less than 50 ppm. 28.Formulation according to claim 27, characterized in that its calciumcontent is less than 20 ppm.
 29. Formulation according to claim 28,characterized in that its calcium content is less than 5 ppm. 30.Process according to claim 13, characterized in that the amount ofcalcium in the liquid pharmaceutical formulation administered to thepatient is less than 20 ppm.
 31. Process according to claim 30,characterized in that the amount of calcium in the liquid pharmaceuticalformulation administered to the patient is less than 5 ppm.